Discovery of the SHP2 allosteric inhibitor 2-((3R,4R)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-(2,3-dichlorophenyl)-3-methylpyrrolo[2,1-f][1,2,4] triazin-4(3H)-one

Abstract The non-receptor protein tyrosine phosphatase (PTP) SHP2 encoded by the PTPN11 gene is a critical regulator in a number of cellular signalling processes and pathways, including the MAPK and the immune-inhibitory programmed cell death PD-L1/PD-1 pathway. Hyperactivation and inactivation of SHP2 is of great therapeutic interest for its association with multiple developmental disorders and cancer-related diseases. In this work, we characterised a potent SHP2 allosteric inhibitor 2-((3 R,4R)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-(2,3-dichlorophenyl)-3-methylpyrrolo[2,1-f][1,2,4]triazin-4(3H)-one (PB17-026-01) by using structure-based design. To study the structure–activity relationship, we compared co-crystal structures of SHP2 bound with PB17-026-01 and its analogue compound PB17-036-01, which is ∼20-fold less active than PB17-026-01, revealing that both of the compounds are bound to SHP2 in the allosteric binding pocket and PB17-026-01 forms more polar contacts with its terminal group. Overall, our results provide new insights into the modes of action of allosteric SHP2 inhibitor and a guide for the design of SHP2 allosteric inhibitor.


Introduction
The non-receptor protein tyrosine phosphatase (PTP) SHP2 encoded by the PTPN11 gene, also named PTP1D or PTP-2C, is the first reported proto oncoprotein of human PTP family that is widely expressed in various adult cells and tissues. 1 The structure of SHP2 consists of three domains: the Src homology (SH) regions, one catalytic PTP domain (residues 247-517), and one C-terminal tail with tyrosyl phosphorylation sites (Y542 and Y580) as well as a proline-rich motif. 2 The SH regions denoted as N-SH2 (residues 6-104) and C-SH2 (residues 112-216) are recognition elements to bind the phosphorylated tyrosine protein sequences. X-ray structures demonstrate that the basal state SHP2 adopts an auto-inhibited conformation, of which the "backside loop" of the N-SH2 domain intramolecularly interacts with the catalytic surface of the PTP domain at the N-SH2: PTP interface to block access of the substrate to the catalytic site. The C-SH2 domain acts as a linker connecting the neighbour N-SH2 and PTP sites. However, neither of them shares a significant interface with C-SH2 domain. The activity of SHP2 is regulated by a mechanism similar to the "molecular switch". Receptor tyrosine kinases (RTKs) are first activated by tyrosine-phosphorylated upstream signal factors (such as IL-2, IL-6, IFN-alpha, and EGF), leading to the distortion of the N-SH2 domain from the PTP domain, which makes the catalytic site accessible to substrates and changes SHP2 to the "on" state.
Consequently, RAS is directly dephosphorylated and associated with the effector protein RAF. Therefore, SHP2 acts as a mediator in cell signal transduction downstream of various RTKs, including the RAS/MAPK/ERK, PI3K/AKT, mTOR, and JAK/STAT pathways. 3 Recently, this ubiquitously expressed enzyme is found to be required for full and sustained participation in the immuneinhibitory reactions including programmed cell death-1 (PD-1) to modulate the activation of T cells. Thus, SHP2 is a fascinating target in immuno-oncology. 4,5 Taken together, SHP2 is involved in the regulation of diverse phase in the cell life such as proliferation, survival, differentiation, migration, and apoptosis. Hyperactivation of SHP2 caused by PTPN11 germline or somatic mutations contributes to oncogenesis and underlies developmental disorders. Active mutations of SHP2 have been identified in Noonan syndrome, 6 juvenile myelomonocytic leukaemia, 7 B-cell acute lymphoblastic leukaemia, myelodysplastic syndrome, acute myeloid leukaemia, and various solid tumours including lung adenocarcinoma, colon cancer, breast cancers, gastric cancer and glioblastoma, neuroblastoma, melanoma, and hepatocellular carcinoma. 8 In LEOPARD syndrome, mutation is located in the SHP2 catalytic domain, which abolishes the SHP2 phosphatase activity. 9 SHP2 is a promising therapeutic target for its functions of regulating the aforementioned signalling pathways and the potential link between diseases and SHP2 mutations.
As an excellent multi-disease target, the discovery of small molecule inhibitors has attracted significant attention in the scientific community. Efforts to discover SHP2-targeted small molecule inhibitors targeting the active site such as NSC87877, 10 II-B08, 11 and NAT6-297775 12 provide insights into the inhibitory mechanism that helps to design more efficient inhibitors. However, these active site inhibitors often lack selectivity because of high protein sequence identity within the inherent catalytic site among all PTPs. Additionally, the solvated, polar, and positive-charged environment of the PTP active pocket requires multiple ionisable functional groups of inhibitors to compete with the substrate. These functional groups, however, cause problems of low potency, poor cell permeability, and oral bioavailability. To overcome the obstacles of active site inhibitors, novel allosteric inhibitors stemming from chemical, mechanism-based, and computer-aided studies focussed on high selectivity and low toxicity are gaining an intensive research momentum recently. Initial medicinal chemistry research identified allosteric inhibitors targeting SHP2 with moderate potency, selectivity, and oral bioavailability, as exemplified by Novartis developed SHP099. 13 SHP099 concurrently inserts into the "blocking" loop located at the interdomain interface of N-SH2, C-SH2, and PTP domains, which stabilises SHP2 in the inactive "closed" conformation. Moreover, SHP099 shows high selectivity over 66 kinases and 21 phosphatases including SHP1, which shares more than 60% identity with SHP2. For now, several allosteric inhibitors have been successively discovered and at least three candidates including Novartis's TNO155, 14 RMC-4630, 15 and JAB-3068 16 are currently undergoing in Phase II clinical trials. Therefore, discovery of novel allosteric inhibitors with diverse chemical backbones is of great importance.

Results and discussion
We first designed a series of compounds (see supporting information for the spectral data including 1 H-NMR and 13 C-NMR about the compounds) derived from SHP099 and their inhibitory activities were measured (Table 1 and Figure 1). Of these compounds, the compound PB17-026-01 showed the highest inhibitory activity with a IC 50 value of 38.9 nM, which is better than SHP099 ( Figure 2).
To investigate how PB17-026-01 interacts with SHP2, we studied the co-crystal structure of SHP2 in complex with PB17-026-01 (PDB-ID: 7XBQ) (see Tables 2 and 3 for detailed information about protein expression and crystallised macromolecule preparation). The crystal structure solved at 2.20 Å resolution provides detailed information about the interaction mode between SHP2 and PB17-026-01. The crystal belongs to the orthorhombic space group P2 1 2 1 2 1 with the unit cell dimensions of a ¼ 55.69 Å, b ¼ 91.39 Å, and c ¼ 212.98 Å ( Table 4). As previously reported, the SHP2 protein contains three potential small molecule modalities: (1) the "tunnel-like" pocket formed by the inter-domain interface of N-SH2, C-SH2, and PTP domains; (2) the "latch" locus located at the interface of N-SH2 and PTP domains, which is approximately 20 Å from the tunnel; (3) the "groove" site on the opposite side of the protein. 17 The electron density clearly reveals that PB17-026-01 perfectly adheres to the central tunnel region (estimated volume ¼ 464 Å 3 ) and interacts with all three domains of SHP2, locking the enzyme in an auto-inhibited, inactive conformation (Supplementary Figure S1A). At the tunnel interface, PB17-026-01 is hydrogen-bonded with N e of R111 in the N-SH2 domain, main chain carboxyl O of F113 in the C-SH2 domain, O b of T253 in the PTP domain, main chain carboxyl O of T108 and E110, respectively ( Figure 3(A)). In addition, the dichlorophenyl moiety of PB17-026-01 occupies an extensive hydrophobic area of the pharmacophore by interacting with L254, Q257, P491, and Q495 in the PTP domain. Compared with SHP099 (Supplementary Figure S2), PB17-026-01 lacks the hydrogen bond with E250 but forms hydrogen bond with T253; meanwhile it is hydrogen-bonded with N e instead of N g of R111 (Figure 3(C)). The binding of PB17-026-01 does not change the conformation of SHP2 significantly and the co-crystal structure is almost identical with the inactive apo SHP2 (PDB code: 2SHP) with the N-SH2 domain sealing the active site. The interaction between the N-SH2 domain and the PTP domain remains in resting state with unchanged relative position of each domain. PB17-021-01 differs from PB17-026-01 only by a replacement of a C atom with a N atom on the five-member ring; however, the former has a decreased inhibition activity (IC 50 ¼ 104.2 nM). Co-crystal structure of SHP2 in complex with PB17-026-01 shows that this C atom is in the vicinity of two hydrophobic residues L254 and P491. There are inherent hydrophobic and desolvation penalties incurred upon the substitution of a C atom with a N atom which leads to the decreased binding activity.
PB17-036-01 is $20-fold less active (IC 50 ¼ 645 nM) than PB17-026-01and the only difference between the two compounds is at the terminal group. To investigate the structure-activity relationship, we examined the co-crystal structure of SHP2 in complex with PB17-036-01 (PDB-ID: 8GWW). The crystal structure was solved at 3.0 Å resolution and the structure clearly reveals that PB17-036-01 perfectly adheres to the central tunnel region resembling PB17-026-01 (Supplementary Figure S1B). Superimposition of the co-crystal structures of PB17-036-01 and the lead inhibitor PB17-026-01 (Figure 3(D)) show that PB17-036-01 is hydrogenbonded with N e of R111 in the N-SH2 domain, main chain carboxyl O of F113, and side-chain carboxyl O of E249 in the same mode as PB17-026-01 (Figure 3(B)). However, the terminal group of PB17-026-01 is hydrogen-bonded with four residues T108, E110, F113, and T253, whereas the terminal group of PB17-036-01 is only hydrogen-bonded with F113 and E249, which explains the improved binding activity of PB17-026-01.

Conclusion
As the central role of SHP2 in developmental and oncogenic diseases, the development of potent, selective, and orally efficacious    SHP2 inhibitors has attracted interest of many pharmaceutical researches and developments but remains a challenge. In this study, we identified a new compound PB17-026-01 derived from SHP099 but with significantly different chemical backbone that allosterically inhibits SHP2 potently. We also solved the co-crystal structures of SHP2 in complex with PB17-026-01 and its analogue compound PB17-036-01, which only differs from PB17-026-01 at the terminal group. These structures indicate that both of PB17-026-01 and PB17-036-01 are inserted in the tunnel pocket to stabilise the auto-inhibited and inactive conformation with different binding mode form SHP099 and PB17-026-01 forms more hydrogen bonds with SHP2 than PB17-036-01 because of their different terminal groups. For its high potency and new backbone structure, the compound provides new insights into SHP2 inhibition and a suitable basis for further development of potent, selective, and orally bioavailable compounds leading to treat SHP2-dependent diseases.

Experimental section
The synthesis of PB17-026-01 A small amount of sodium hydride (NaH, 130 mM) was added to methyl 3-bromopyrrole-2-carboxylate (100 mM) in DMF (100 mL) multiple times. The reaction mixture was stirred at 0 C for 20 min, followed by the addition of the O-diphenylphosphinylhydroxylamine (120 mM). The mixture was incubated at room temperature

Protein expression and purification
The gene sequence encoding SHP2 residues Met1-Leu525 (UniProt accession code Q06124) was synthesised by GenScript (GenScript.com) with codon optimisation for E. coli expression and inserted into pET-15b containing an N-Terminal 6 Â His-tag and a tobacco etch virus (TEV) cleavage site. The E. coli BL21 (DE3) cells transformed with the recombinant plasmid were grown in Luria-Bertani medium at 37 C until OD 600 reached 0.6-0.8. Protein expression was induced by isopropyl-b-D-thiogalactoside to a final concentration of 0.2 mM and the cells were grown overnight at 18 C and then harvested by centrifugation at 6000g for 15 min at 4 C. The cell pellet was re-suspended in buffer A (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5% glycerol) and lysed by a high-pressure homogeniser at 1000 bar. Cell debris was removed by centrifugation at 47,000g for 30 min. The supernatant was loaded onto a 5 mL Ni-NTA column (GE Healthcare), which had been equilibrated in buffer A. A gradient wash was applied with increasing concentration of imidazole (10-50 mM) in the same buffer in order to remove non-specific bound protein. The His-tagged SHP2 was eluted with 250 mM imidazole in the same buffer. The 6 Â His-tag was cleaved by TEV protease overnight at 4 C. The TEV protease and uncleaved 6 Â His-tagged protein were removed by passing through the Ni-NTA column again. The flow-through containing His-tag removed SHP2 was concentrated and further purified by a size-exclusion column Superose 12 10/300 (GE Healthcare) and the purest fraction of SHP2 was concentrated to $12 mg/mL in 5 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 5 mM DTT.

Crystallisation and structure determination
Crystallisation was performed in MRC 96-well two-drop sitting drop plates by using the sitting-drop vapour-diffusion method. Half microliter of protein solution containing 12 mg/mL in 5 mM Tris-HCl pH 8.0, 100 mM NaCl, and 5 mM DTT was mixed with 0.5 lL of crystallisation well solution containing 13-18% PEG 4000, and 0.1 M Tris-HCl pH 8.5. The crystals appeared after a few hours and reached the maximum size after 24 h. Then the crystals were transferred to a new drop containing 18% PEG 4000, and 0.1 M Tris-HCl pH 8.5 and the inhibitor powders were added to the drop to soak the crystals. The SHP2-inhibitor complex crystals were transferred to a cryoprotectant solution comprising 18% PEG 4000, 0.1 M Tris-HCl pH 8.5 and 20% glycerol, and then flash frozen in liquid nitrogen. Diffraction data were collected from a single crystal at 100 K at BL19U1 of the Shanghai Synchrotron Radiation Facility. The reflections were indexed, integrated, and processed with iMosflm. 18 The space group of the complex was P2 1 2 1 2 1 with two molecules in the asymmetric unit. The structure was solved by molecular replacement implemented in Phaser 19 using the SHP2 structure (PDB code 6WU8) as the original search model. The structure model of the inhibitor was manually fit in the electron density using Coot. 20 Structure refinements were performed by using Phenix 21 ( Table 5) and figures of the models were created with PYMOL (The PyMOL Molecular Graphics System, Schr€ odinger, LLC).

Measurement of phosphatase activity
The auto-inhibition conformation of SHP2 is allosterically activated by the binding of bis-tyrosyl-phosphorylated peptides, which makes the PTP domain of SHP2 active and available for substrate recognition and subsequent reaction catalysis. The measurement of the catalytic activity of SHP2 is using the surrogate substrate 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) in a prompt fluorescence assay format. 14,22,23 In special, the phosphatase reaction was performed at room temperature in a 384-well black plate (Corning) with a total volume of 20 lL. The assay buffer contained 60 mM HEPES, pH7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 5 mM DTT, and 0.05% Triton X-100. The inhibition of SHP2 from the 5 lL tested compound was monitored using an assay in which 0.5 nM of SHP2 was incubated with of 0.125 mM of bis-tyrosyl-phosphorylated peptide. After 60 min incubation at room temperature, the surrogate substrate DiFMUP (Thermo, Cat: D6567, 200 lM) was added to the catalytic reaction. After 30 min incubation at room temperature, the fluorescence signal was measured at the excitation and emission wavelengths of 340 nm and 450 nm respectively with a plate reader (Tecan Spark). The curves of the inhibitor dose-response were analysed using normalised IC 50 regression curve fitting with control-based normalisation.